Imer, 59ATACACTGGCCCGAGGCAAC39; reverse primer, 59CCACATCTCGGATCATGCTTTC39; IL-1b: forward primer, 59CTACCTATGTCTTGCCCGTGGAG39; reverse primer, 59GGGAACATCACACACTAGCAGGTC39; IL-6: forward primer, 59ATTGTATGAACAGCGATGATGCAC39; reverse primer, 59CCAGGTAGAAACGGAACTCCAGA39; GAPDH: forward primer, 59GGCACAGTCAAGGCTGAGAATG39; reverse primer, 59ATGGTGGTGAAGACGCCAGTA39. These primers were purchased from Takara Bio (Shiga, Japan). Realtime PCR was performed using the ABI prism 7500 Sequence Detection System (Applied Biosystems, Foster City, Calif.). All data ware subsequently normalized to the glyceraldehyde-3phosphate dehydrogenase (GAPDH) mRNA level and expressed as mRNA relative fold change.Intracerebroventricular InjectionAt 10 day after MI or sham operation, we divided rats into two groups, treated with ICV injection of TLR4-SiRNA or hGAPDHSiRNA for 2 weeks. Prior at 10 day after coronary ligation, we could not do ICV infusion safely because the surgical procedures of the ICV infusion, involving anesthesia, are too hard for rats with MI-induced heart failure. In addition, we did ICV infusion again at 17 day after MI or sham operation. The rats were anesthetized with sodium pentobarbital (50 mg/kg intraperitoneally) and placed in stereotaxic instrument, and a small hole was drilled in the skull for ICV injection of TLR4-SiRNA using glass microsyringe in the right Title Loaded From File lateral ventricle (1.5 mm lateral and 1 mm posterior to the bregma, 3.8 mm in depth). 10 mg SiRNA dissolved in 10 ml water was administered for 5 minutes.Hemodynamics MeasurementsTo evaluation of LVDS, LVDD, LVEF, and FS, cardiac echocardiography was performed at the end of the protocol under light sodium pentobarbital anesthesia (50 mg/kg intraperitoneally) with spontaneous respiration. An echocardiography system (SSD5000; Aloka, Tokyo, Japan) with a Title Loaded From File dynamically focused 7.5 MHz linear array transducer was used. LVDD amd LVDS were obtained in M-mode tracings from the short-axis view at the level of the papillary muscle. LV end-diastolic volume (LVEDV) and LV end-systolic volume (LVESV) 18055761 were obtained in twodimensional mode by taking the measurement of short-axis cross sectional areas (A) and LV length (L) (LV volume = 5/6AL, diastolic and systolic separately) [32]. LVEF was calculated by the following formula: LVEF = (LVEDV-LVESV)/LVEDV6100 . FS was calculated as percentage in accordance with the following formula: FS = (LVDD-LVDS)/LVDD6100( ). To measure mean BP (mBP), LVEDP, LV dP/dtmax, LV -dP/ dtmax, and heart rate, at the end of the protocol, rats were anesthetized with sodium pentobarbital (50 mg/kg intraperitone-StatisticsData are expressed as mean 6 SEM. The statistical analyses were performed by nonpaired t test when comparing data between the 2 groups. A one-way analysis of variance (ANOVA) for the multiple group of ICV injection of SiRNA experiments was performed. Differences were considered to be statistically significant at a P value of ,0.05.Author ContributionsConceived and designed the experiments: KO YH TK KS. Performed the experiments: KO TK. Analyzed the data: KO YH TK TI. Contributed reagents/materials/analysis tools: KO YH TK. Wrote the paper: KO YH TK.HIV-1 infection is a chronic infection with non-stop viral replication leading to a decrease in the number of TCD4 lymphocytes and immunodepression. Viral replication can be limited by antiretroviral drugs of different classes. This reduction in viral replication, which is generally below the threshold of the vi.Imer, 59ATACACTGGCCCGAGGCAAC39; reverse primer, 59CCACATCTCGGATCATGCTTTC39; IL-1b: forward primer, 59CTACCTATGTCTTGCCCGTGGAG39; reverse primer, 59GGGAACATCACACACTAGCAGGTC39; IL-6: forward primer, 59ATTGTATGAACAGCGATGATGCAC39; reverse primer, 59CCAGGTAGAAACGGAACTCCAGA39; GAPDH: forward primer, 59GGCACAGTCAAGGCTGAGAATG39; reverse primer, 59ATGGTGGTGAAGACGCCAGTA39. These primers were purchased from Takara Bio (Shiga, Japan). Realtime PCR was performed using the ABI prism 7500 Sequence Detection System (Applied Biosystems, Foster City, Calif.). All data ware subsequently normalized to the glyceraldehyde-3phosphate dehydrogenase (GAPDH) mRNA level and expressed as mRNA relative fold change.Intracerebroventricular InjectionAt 10 day after MI or sham operation, we divided rats into two groups, treated with ICV injection of TLR4-SiRNA or hGAPDHSiRNA for 2 weeks. Prior at 10 day after coronary ligation, we could not do ICV infusion safely because the surgical procedures of the ICV infusion, involving anesthesia, are too hard for rats with MI-induced heart failure. In addition, we did ICV infusion again at 17 day after MI or sham operation. The rats were anesthetized with sodium pentobarbital (50 mg/kg intraperitoneally) and placed in stereotaxic instrument, and a small hole was drilled in the skull for ICV injection of TLR4-SiRNA using glass microsyringe in the right lateral ventricle (1.5 mm lateral and 1 mm posterior to the bregma, 3.8 mm in depth). 10 mg SiRNA dissolved in 10 ml water was administered for 5 minutes.Hemodynamics MeasurementsTo evaluation of LVDS, LVDD, LVEF, and FS, cardiac echocardiography was performed at the end of the protocol under light sodium pentobarbital anesthesia (50 mg/kg intraperitoneally) with spontaneous respiration. An echocardiography system (SSD5000; Aloka, Tokyo, Japan) with a dynamically focused 7.5 MHz linear array transducer was used. LVDD amd LVDS were obtained in M-mode tracings from the short-axis view at the level of the papillary muscle. LV end-diastolic volume (LVEDV) and LV end-systolic volume (LVESV) 18055761 were obtained in twodimensional mode by taking the measurement of short-axis cross sectional areas (A) and LV length (L) (LV volume = 5/6AL, diastolic and systolic separately) [32]. LVEF was calculated by the following formula: LVEF = (LVEDV-LVESV)/LVEDV6100 . FS was calculated as percentage in accordance with the following formula: FS = (LVDD-LVDS)/LVDD6100( ). To measure mean BP (mBP), LVEDP, LV dP/dtmax, LV -dP/ dtmax, and heart rate, at the end of the protocol, rats were anesthetized with sodium pentobarbital (50 mg/kg intraperitone-StatisticsData are expressed as mean 6 SEM. The statistical analyses were performed by nonpaired t test when comparing data between the 2 groups. A one-way analysis of variance (ANOVA) for the multiple group of ICV injection of SiRNA experiments was performed. Differences were considered to be statistically significant at a P value of ,0.05.Author ContributionsConceived and designed the experiments: KO YH TK KS. Performed the experiments: KO TK. Analyzed the data: KO YH TK TI. Contributed reagents/materials/analysis tools: KO YH TK. Wrote the paper: KO YH TK.HIV-1 infection is a chronic infection with non-stop viral replication leading to a decrease in the number of TCD4 lymphocytes and immunodepression. Viral replication can be limited by antiretroviral drugs of different classes. This reduction in viral replication, which is generally below the threshold of the vi.